The invention relates to compounds, compositions, and methods for blocking protein-protein interactions.
Many pharmaceutical drugs act by blocking the binding of an enzyme to its substrate. In order for these drugs to block this interaction effectively, they must bind to the target enzymes more tightly than the substrate binds. Binding strengths are determined in part by the number of favorable contacts between the compounds. Since most enzyme substrates are small molecules, small molecule drugs can be engineered to make as many (or, if desired, more) contacts with the enzyme than does the substrate, facilitating tight enzyme interactions. As an illustration, FIG. 1A shows an enzyme 1 bound to its substrate 2. FIG. 1B shows the enzyme 1 bound to a small molecule drug 3. In addition, because enzymes work by lowering the energy of the transition state between substrate and product, the enzyme binds to especially tightly to transition state analogues. Drugs that resemble the transition state can bind more tightly to an enzyme than does the normal substrate, again affording an opportunity for antagonist-type drug design.
In contrast, it is generally more difficult to engineer small molecule compounds that block the interaction of a target protein with another protein, because interacting proteins usually contact each other over large surface areas and make many favorable contacts. It is therefore difficult for a small molecule to have a greater number of favorable contacts with a protein than does another protein. As an illustration, FIG. 1C shows two such interacting proteins, 4 and 5, and indicates the large number of favorable contacts between these proteins. In addition, interacting proteins do not possess transition states analogous to those exhibited by substrates and their products. Thus, there is no specialized conformation that a drug can mimic to bind more effectively to a target protein.
There are many instances in which it is therapeutically useful to block the interaction of a target protein with another protein. Examples of biological events that involve protein-protein interactions include signal transduction, transcription, protein ligand-receptor interactions, and protein assembly.
The ability to block these processes specifically facilitates the development of therapies for diseases that are currently difficult to treat. Accordingly, compounds that block interactions such as those described above represent potentially useful drugs for treating, preventing, or reducing the severity of certain diseases or their symptoms. For example, viral infections (such as herpes, hepatitis C, HIV, and influenza infections) could be treated with compounds that block the assembly of viral proteins, or with compounds that prevent the ligand-receptor interaction of a virus attaching to a host cell.
The invention features a compound having a molecular weight of less than 1500 daltons that non-covalently interacts with, and covalently bonds to, a target protein at an amino acid side chain that is not part of an enzyme active site; a covalently bound portion of the compound sterically blocks the binding of the target protein to a second protein. The compound bonds to the target protein with a forward reaction rate that is at least 100 times faster than the forward reaction rate at which the compound bonds to the side chain of the corresponding free amino acid under physiological conditions. The compound may bond in such a way that essentially all, most, or only a small portion, of the compound remains covalently attached to the target protein; another portion of the compound may serve as a leaving group. For example, in some instances, only an acyl group, preferably an acetyl group, remains attached to the target protein.
A preferred compound bonds to the target protein with a forward reaction rate that is at least 1000 times faster than the forward reaction rate at which the compound bonds to the side chain of the free amino acid under physiological conditions, and more preferably bonds with a rate 10,000 times, or 100,000 times faster. The compound is preferably a synthetic compound.
In addition, a preferred compound has a covalent bonding rate constant with the side chain of the free amino acid of less than 10xe2x88x925/M/sec at room temperature under physiological conditions, and more preferably has a rate constant of less than 10xe2x88x926/M/sec, or 10xe2x88x927/M/sec at room temperature under physiological conditions.
The non-specific covalent bonding rate constant for penicillin to form stable bonds with amino acid side chains, such as those of serine and lysine, is about 8xc3x9710xe2x88x926/M/sec. Because penicillin is a useful drug whose side effects, which are results of its reactivity, are considered to be acceptable, it is expected that the side effects resulting from the non-specific reactivity of drugs with similar or smaller covalent bonding rate constants will also be acceptable.
A preferred compound also includes a Specificity Group whose removal results in the bonding reaction rate with the side chain of the corresponding free amino acid being substantially unchanged, and the bonding reaction rate with the target protein being reduced to a rate that is substantially similar to the bonding reaction rate with the side chain of the free amino acid; the compound also includes a Bonding Group that forms the covalent bond with the target protein. In this compound, modification of the Specificity Group does not substantially alter the bonding reactivity of the Bonding Group. In such preferred compounds, the Bonding Group and Specificity Group are connected by appropriate linkers, so that, for example, electronic effects are not transmitted from the Specificity Group to the Bonding Group. An example of an appropriate linker is an alkyl chain having 2-12 carbon atoms.
Preferred target proteins include kinases, viral coat proteins, STAT proteins, oncogenes, transcription factors, and extracellular protein ligands, protein domains, and their receptors. More specifically, preferred target proteins include MCP-1, Fos, and IL-1 beta. In a preferred compound, the Bonding Group forms a covalent bond with the side chain of the amino acid of the target protein as a result of the intrinsic reactivity of the Bonding Group.
The purpose of the Specificity Group is to direct the compound to a particular protein target and to position the Bonding Group near an amino acid side chain on the target protein; the Bonding Group will therefore have a high effective concentration, relative to the amino acid side chain, and will react with the side chain. An initial Specificity Group may be obtained by screening conventional chemical compound libraries for compounds that non-covalently interact with the target protein. Alternatively, the initial Specificity Group could be obtained by rational drug design, or by using a peptide that is known to bind to the target protein.
For comparison purposes, the side chain of the free amino acid that corresponds to the amino acid that forms a covalent bond to the B group is used, rather than a side chain on a non-target protein. The side chain of the corresponding free amino acid can always be clearly defined; in addition, it will not be subject to environmental influences, such as steric factors, that may vary from one non-target protein to the next.
It is useful, during the improvement of Bifunctional Blockers that is described in detail below, for the Bonding Group and Specificity Group to be connected so that modification of one Group has little effect on the other. In brief, improvement of Bifunctional Blockers is accomplished by systematically improving the Specificity Group and weakening the reactivity of the Bonding Group. There is an extensive body of knowledge, well known to those skilled in the art of organic chemistry, that predicts the relative reactivity of possible Bonding Groups. This knowledge can be used to systematically alter and weaken a Bonding Group during the course of improvement of a Bifunctional Blocker. However, if the Specificity Group is connected to the Bonding Group in such a way that the Specificity Group""s composition influences the Bonding Group""s reactivity, then it will be difficult to systematically weaken the reactivity of the Bonding Group. It is therefore preferable to connect these Groups so that they do not influence each other.
The invention also features a compound having a molecular weight of less than 1500 daltons that covalently bonds to an amino acid side chain of a target protein that is not an enzyme; a covalently bound portion of the compound sterically blocks the binding of the target protein to a second protein. The compound bonds to the target protein with a forward reaction rate that is at least 100 times faster than the forward reaction rate at which the compound bonds to the side chain of the corresponding free amino acid under physiological conditions. A preferred compound bonds to the target protein with a forward reaction rate that is at least 1000 times faster than the forward reaction rate at which the compound bonds to the side chain of the free amino acid under physiological conditions, and more preferably bonds with a rate 10,000 times, or 100,000 times faster.
In this case, a preferred compound also has a covalent bonding rate constant with the side chain of the free amino acid of less than 10xe2x88x925/M/sec at room temperature under physiological conditions, and more preferably has a rate constant of less than 10xe2x88x926/M/sec, or 10xe2x88x927/M/sec at room temperature under physiological conditions. A preferred compound is one that is prepared synthetically.
In addition, a preferred compound includes a Specificity Group whose removal results in the bonding reaction rate with the side chain of the free amino acid being substantially unchanged, and the bonding reaction rate with the target protein being reduced to a rate that is substantially similar to the bonding reaction rate with the side chain of the free amino acid; the compound also includes a Bonding Group that forms the covalent bond with the target protein. In this compound, modification of the Specificity Group does not substantially alter the bonding reactivity of the Bonding Group. Preferred target proteins include kinases, viral coat proteins, STAT proteins, oncogenes, transcription factors, and extracellular protein ligands, protein domains, and their receptors. More specifically, preferred target proteins include MCP-1, Fos, and IL-1 beta.
The invention also features a substantially pure compound having a molecular weight of less than 1500 daltons that includes a Specificity Group that non-covalently interacts with the surface of a target protein and a Bonding Group that covalently bonds to the target protein. A covalently bound portion of the compound competitively and sterically inhibits the binding of a second protein to the target protein.
The invention further features a combinatorial chemistry library containing at least 25 compounds. Each compound contains the same Bonding Group, which covalently bonds to the side chains of amino acids. The covalent bonding rate constant is between 10xe2x88x923/M/sec and 10xe2x88x927/M/sec for the reaction of the Bonding Group with the side chain of the free amino acid that reacts most rapidly with the Bonding Group to give a stable product (i.e., a product that has a sufficiently long half-life in physiological conditions to inhibit the target protein usefully). In preferred libraries, each compound also includes a Specificity Group.
The invention further features a pharmaceutical composition including a compound having a molecular weight of less than 1500 daltons; the compound non-covalently interacts with, and covalently bonds to, a target protein at an amino acid side chain that is not part of an enzyme active site. A covalently bound portion of the compound sterically blocks the binding of the target protein to a second protein. The compound bonds to the target protein with a forward reaction rate that is at least 100 times faster than the forward reaction rate at which the compound bonds to the side chain of the corresponding free amino acid under physiological conditions. The pharmaceutical composition also includes a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention are free of contaminating substances, such as trace organic solvents and synthetic precursors, that would make the composition unacceptable for clinical use.
In a preferred composition, the compound bonds to the target protein with a forward reaction rate that is at least 1000 times faster than the forward reaction rate at which the compound bonds to the side chain of the free amino acid under physiological conditions, and more preferably bonds with a rate 10,000 times, or 100,000 times faster. In another preferred composition, the compound has a covalent bonding rate constant with the side chain of the free amino acid of less than 10xe2x88x925/M/sec at room temperature under physiological conditions, and more preferably has a rate constant of less than 10xe2x88x926/M/sec, or 10xe2x88x927/M/sec at room temperature under physiological conditions.
Another preferred composition includes a compound that includes a Specificity Group whose removal results in the bonding reaction rate with the side chain of the free amino acid being substantially unchanged, and the bonding reaction rate with the target protein being reduced to a rate that is substantially similar to the bonding reaction rate with the side chain of the free amino acid. In this composition, the compound includes a Bonding Group that forms the covalent bond with the target protein. Modification of the Specificity Group does not substantially alter the bonding reactivity of the Bonding Group. Preferred target proteins include kinases, viral coat proteins, STAT proteins, oncogenes, transcription factors, and extracellular protein ligands and their receptors. More specifically, preferred target proteins include MCP-1, Fos, and IL-1 beta. In a preferred composition, the Bonding Group forms a covalent bond with the amino acid side chain of the target protein as a result of the Bonding Group""s intrinsic reactivity.
The invention also features a composition containing a compound having a molecular weight of less than 1500 daltons that non-covalently interacts with, and covalently bonds to, a target protein that is not an enzyme; a covalently bound portion of the compound sterically blocks the binding of the target protein to a second protein. The compound bonds to the target protein with a forward reaction rate that is at least 100 times faster than the forward reaction rate at which the compound bonds to the side chain of the corresponding free amino acid under physiological conditions. A preferred composition includes a compound that bonds to the target protein with a forward reaction rate that is at least 1000 times faster than the forward reaction rate at which the compound bonds to the side chain of the free amino acid under physiological conditions, and more preferably bonds with a rate 10,000 times, or 100,000 times faster.
In another preferred composition, the compound has a covalent bonding rate constant with the side chain of the free amino acid of less than 10xe2x88x925/M/sec at room temperature under physiological conditions, and more preferably has a rate constant of less than 10xe2x88x926/M/sec, or 10xe2x88x927/M/sec at room temperature under physiological conditions.
In addition, a preferred composition contains a compound that includes a Specificity Group whose removal results in the bonding reaction rate with the side chain of the corresponding free amino acid being substantially unchanged, and the bonding reaction rate with the target protein being reduced to a rate that is substantially similar to the bonding reaction rate with the side chain of the free amino acid; this compound also includes a Bonding Group that forms the covalent bond with the target protein. Modification of the Specificity Group does not substantially alter the bonding reactivity of the Bonding Group. Preferred target proteins include kinases, viral coat proteins, STAT proteins, oncogenes, transcription factors, and extracellular protein ligands, protein domains, and their receptors. More specifically, preferred target proteins include MCP-1, Fos, and IL-1 beta.
The invention further features a method of producing a compound that selectively binds to a target protein. The method includes (a) starting with a candidate compound having a molecular weight of less than 1500 daltons and including a first group and a second group, where the second group covalently bonds to an amino acid side chain of the target protein and the first group does not covalently bond to any amino acid side chain, but may non-covalently interact with the target protein; (b) testing the candidate compound for inhibition of the interaction of the target protein and a second protein; (c) testing the candidate compound for binding to a non-target molecule, such as an amino acid or non-target protein; (d) testing the candidate compound for bonding reactivity of the second group with the amino acid side chain of the target protein; (e) altering the first group and selecting for increased selectivity for the target protein, where increased selectivity is indicated by an increase in inhibition of the interaction of the target protein and a second protein, relative to binding to the non-target molecule; (f) altering the second group and selecting for decreased bonding reactivity of the second group with the amino acid side chain of the target protein and with the non-target molecule; and (g) optionally repeating steps (e) and (f), to obtain a compound that has selectivity for the target protein, relative to the non-target molecule, at least 100 times greater than the candidate compound of step (a).
Preferably, the compound selectively binds to the target protein at an amino acid side chain that is not part of an enzyme active site, and also binds to the target protein at a site of protein-protein interaction. In a preferred method, the candidate compound is present in or chosen from a combinatorial chemistry library. In a different preferred method, the compound is designed by principles of rational drug design or with the aid of a computer molecular modeling system. In another preferred method, the first group is a Specificity Group that non-covalently binds to the protein under physiological conditions, the second group is a Bonding Group that forms a covalent bond with the amino acid side chain on the protein, and modification of the Specificity Group does not substantially alter the reactivity of the Bonding Group. In still another preferred method, the candidate compound of step (a) is a compound having a molecular weight of less than 1500 daltons that covalently bonds to a target protein at an amino acid side chain that is not part of an enzyme active site; a covalently bound portion of the compound sterically blocks the binding of the target protein to a second protein. The candidate compound bonds to the target protein with a forward reaction rate that is at least 100 times faster than the forward reaction rate at which the compound bonds to the side chain of the corresponding free amino acid under physiological conditions.
The invention also features a method of producing a compound that selectively binds to a target protein; the method includes (a) starting with a candidate compound having a molecular weight of less than 1500 daltons and including a first group and a second group, where the second group covalently bonds to an amino acid side chain of the target protein and the first group does not covalently bond to any amino acid side chain, but may non-covalently interact with the target protein; (b) testing the candidate compound for inhibition of the interaction of the target protein and a second protein; (c) testing the candidate compound for binding to a non-target molecule, such as an amino acid or non-target protein; (d) testing the candidate compound for bonding reactivity of the second group with the amino acid side chain of the target protein; (e) altering the first group and selecting for increased selectivity for the target protein, where increased selectivity is indicated by an increase in inhibition of the interaction of the target protein and a second protein, relative to binding to the non-target molecule; (f) altering the second group and selecting for decreased bonding reactivity of the second group with the amino acid side chain of the target protein and with the non-target molecule; and (g) optionally repeating steps (e) and (f) to obtain a compound that has a selectivity for the target protein, relative to the non-target molecule, at least 100 times greater than the candidate compound of step (a) and that has a covalent bonding rate with the side chain of the corresponding free amino acid of less than 10xe2x88x925/M/sec at room temperature under physiological conditions. Preferably, the compound selectively binds to the target protein at an amino acid side chain that is not part of an enzyme active site, and also binds to the target protein at a site of protein-protein interaction.
The invention also features a method of producing a compound that selectively binds to a target protein including (a) starting with a candidate compound having a molecular weight of less than 1500 daltons and including a first group and a second group, where the second group covalently bonds to an amino acid side chain of the target protein and the first group does not covalently bond to any amino acid side chain, but may non-covalently interact with the target protein; (b) testing the candidate compound for inhibition of the interaction of the target protein and a second protein; (c) testing the candidate compound for binding to a non-target molecule, such as an amino acid or non-target protein; (d) altering the first group and selecting for increased selectivity for the target protein, where increased selectivity is indicated by an increase in inhibition of the interaction of the target protein and a second protein, relative to binding to the non-target molecule; (e) replacing the second group with a group known to be less reactive, altering the first group, and selecting for substantially unchanged or increased inhibition; and (f) optionally repeating steps (d) and (e), to obtain a compound that has a selectivity for the target protein, relative to the non-target molecule, at least 100 times greater than the candidate compound of step (a). Preferably, the compound selectively binds to the target protein at an amino acid side chain that is not part of an enzyme active site, and also binds to the target protein at a site of protein-protein interaction.
Finally, the invention features a method of sterically blocking protein-protein binding. The method includes contacting a target protein with a compound having a molecular weight of less than 1500 daltons, where the compound non-covalently interacts with, and covalently bonds to, the target protein at an amino acid side chain that is not part of an enzyme active site; a covalently bound portion of the compound sterically blocks the binding of the target protein to a second protein. In addition, the compound bonds to the target protein with a forward reaction rate that is at least 100 times faster than the forward reaction rate at which the compound bonds to the side chain of the corresponding free amino acid. The compound has a covalent bonding rate constant with the side chain of the free amino acid of less than 10xe2x88x925/M/sec under physiological conditions. Preferred methods include those used for therapeutic or diagnostic purposes. Additional methods include those used for pesticidal or herbicidal purposes.
In preferred methods, the compounds bonds to the target protein with a forward reaction rate 1000 times faster than the rate at which the compound bonds to the side chain of the corresponding free amino acid, or 10,000, or 100,000 times faster. In other preferred methods, the compound has a covalent bonding rate constant with the side chain of the free amino acid of less than 10xe2x88x926/M/sec or 10xe2x88x927/M/sec under physiological conditions. In addition, in preferred methods, the compound has a Specificity Group and a Bonding Group, and modification of the Specificity Group does not substantially alter the reactivity of the Bonding Group. Preferably, this Bonding Group forms a covalent bond with the side chain of an amino acid of the target protein as a result of the intrinsic reactivity of the Bonding Group.
Preferably, the target protein is not an enzyme and/or the compound is a synthetic compound. Protein-protein binding can be blocked in a mammal, such a human. In preferred instances, the protein-protein binding that is blocked with this method causes a disease.
By a xe2x80x9cSpecificity Groupxe2x80x9d or xe2x80x9cS groupxe2x80x9d is meant a moiety that non-covalently interacts with a target protein, causing a Bonding Group to have a high local concentration near an amino acid side chain on the target protein. The formation of a covalent bond between the Bonding Group and the side chain of the amino acid is accelerated.
An important characteristic of the Specificity Group is that its removal results in the bonding reaction rate with the side chain of an amino acid of a target protein being substantially reduced, and the bonding rate with the corresponding free amino acid being substantially unchanged. Alternatively, removal of the Specificity Group from the compound results in the bonding reaction rate with the side chain of the free amino acid being substantially unchanged, and the bonding reaction rate with the amino acid side chain of the target protein being reduced to a rate that is substantially similar to the bonding reaction rate with that of the free amino acid.
By a xe2x80x9cBonding Groupxe2x80x9d or xe2x80x9cB groupxe2x80x9d is meant a moiety of a compound that forms a covalent bond with an amino acid of a target protein. The Bonding Group does not substantially affect the non-covalent binding affinity of the compound for the target protein.
By xe2x80x9ccorresponding free amino acidxe2x80x9d is meant a free amino acid that is the same amino acid as the amino acid of a target protein that forms a covalent bond with a compound of the invention.
By xe2x80x9cproteinxe2x80x9d is meant a polypeptide having at least 40 amino acids.
By xe2x80x9cBifunctional Blockerxe2x80x9d is meant a compound that has an S group and a B group, that reacts selectively with a target protein, and in which a covalently bound portion of the compound sterically hinders the interaction of the target protein with another protein.
By xe2x80x9croom temperaturexe2x80x9d is meant 25xc2x0 C.xc2x15xc2x0 C.
By xe2x80x9cphysiological solution conditionsxe2x80x9d are meant the conditions, e.g., temperature, pH, and concentration, that are present in the body of the treated organism, such as a human being or other animal, or plant.
By xe2x80x9cstandard lead-optimization proceduresxe2x80x9d are meant processes for drug development that begin with the identification of a lead compound in one or more assays for drug activity. The identification is followed by the synthesis of chemical variants of the lead compound, the testing of the variants in the assays, and the selection of compounds with improved activity and selectivity. The steps of synthesis, testing, and selection are often repeated for several cycles.
By xe2x80x9creactivexe2x80x9d is meant capable of undergoing covalent bond formation or cleavage on a time scale, at concentrations, and under conditions that are relevant to pharmaceutical development, or under physiological conditions.
By xe2x80x9creactive groupxe2x80x9d is meant a chemical moiety that is reactive as a result of its intrinsic chemical properties, and that reacts without the action of enzymes, photons, or molecules that are not present under physiological conditions.
By xe2x80x9cintrinsic reactivityxe2x80x9d is meant the ability to undergo covalent bond formation or cleavage without the action of enzymes, photons, or molecules that are not present under physiological conditions.
By xe2x80x9cnon-target moleculexe2x80x9d is meant any molecule that is used for purposes of comparison with a target molecule in examining the reactivity of a reactive compound. An example of a non-target molecule is a free amino acid to which a reactive compound bonds; another example is a protein, other than the target protein, that contains the same amino acid in a solvent-accessible conformation.
By xe2x80x9cprotein-protein interactionxe2x80x9d is meant contact between proteins that primarily involves polypeptide regions, rather than moieties such as sugars or lipids that are attached to the surface of a protein.
By xe2x80x9csyntheticxe2x80x9d is meant a compound or composition made by, e.g., recombinant techniques or synthetic organic techniques, rather than a compound isolated from a natural source.
By xe2x80x9csubstantially purexe2x80x9d is meant that a compound, such as a Bifunctional Blocker of the invention, has been separated from substances which naturally accompany it, or which are generated during its preparation or extraction. Such substances include organic solvents, reaction precursors, and other possible contaminants. Substances such as water, buffers, chemicals introduced to increase the stability of the compound, or chemicals added for formulation purposes are not considered contaminants. Preferably the compound is at least 80%, more preferably at least 95%, and most preferably at least 99%, by weight, free from other compounds, such as proteins, lipids, and other naturally-occurring molecules with which it is naturally associated. The purity of the compounds of the invention can be measured by any appropriate method, for example, gas chromatography analysis or high performance liquid chromatography analysis.
By a xe2x80x9cdiseasexe2x80x9d is meant a condition of a living organism which impairs normal functioning of the organism, or an organ or tissue thereof.
The present invention provides a number of advantages. For example, the compounds of the invention bind to a non-enzymatic protein or to a surface of an enzyme that is not part of its active site; a covalently bound portion of the compound sterically blocks the binding of that protein to another protein. These compounds contain two distinct chemical moieties: a Specificity Group (S group) that mediates non-covalent interactions with a target protein, and a Bonding Group (B group) that covalently bonds with an amino acid on the target protein. Modification of the S group has a relatively small effect on the bonding activity of the B group, and modification of the B group has a relatively small effect on the non-covalent binding affinity of the S group. As a result of these unique features, the compounds of the invention covalently bond to target proteins tightly enough to result in therapeutic utility, while at the same time bonding to non-target molecules in the body at a rate low enough to avoid drug allergies and other side effects that may result from non-specific interactions with non-target molecules. The methods of the invention provide an improvement over existing drug optimization techniques, in that they allow the independent optimization of distinct portions of compounds of the invention.
Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.